Compositions and methods for treatment of hepatitis C virus-associated diseases

ABSTRACT

Antisense oligonucleotides are provided which are complementary to and hybridizable with at least a portion of HCV RNA and which are capable of inhibiting the function of the HCV RNA. These oligonucleotides can be administered to inhibit the activity of Hepatitis C virus in vivo or in vitro. These compounds can be used either prophylactically or therapeutically to reduce the severity of diseases associated with Hepatitis C virus, and for diagnosis and detection of HCV and HCV-associated diseases. Methods of using these compounds are also disclosed.

INTRODUCTION

This application is a continuation-in-part of U.S. Ser. No. 08/650,093,filed May 17, 1996, which is a continuation-in-part of U.S. Ser. No.08/452,841, filed May 30, 1995, which in turn is a continuation-in-partof U.S. Ser. No. 08/397,220, filed Mar. 9, 1995, which is acontinuation-in-part of U.S. Ser. No. 07/945,289, filed Sep. 10, 1992,now abandoned.

FIELD OF THE INVENTION

This invention relates to the design and synthesis of antisenseoligonucleotides which can be administered to inhibit the activity ofHepatitis C virus in vivo or in vitro and to prevent or treat HepatitisC virus-associated disease. These compounds can be used eitherprophylactically or therapeutically to reduce the severity of diseasesassociated with Hepatitis C virus. These compounds can also be used fordetection of Hepatitis C virus and diagnosis of Hepatitis Cvirus-associated diseases. Oligonucleotides which are specificallyhybridizable with Hepatitis C virus RNA targets and are capable ofinhibiting the function of these RNA targets are disclosed. Methods ofusing these compounds are also disclosed.

BACKGROUND OF THE INVENTION

The predominant form of hepatitis currently resulting from transfusionsis not related to the previously characterized Hepatitis A virus orHepatitis B virus and has, consequently, been referred to as Non-A,Non-B Hepatitis (NANBH). NANBH currently accounts for over 90% of casesof post-transfusion hepatitis. Estimates of the frequency of NANBH intransfusion recipients range from 5%-13% for those receiving volunteerblood, or 25%-54% for those receiving blood from commercial sources.

Acute NANBH, while often less severe than acute disease caused byHepatitis A or Hepatitis B viruses, can lead to severe or fulminanthepatitis. Of greater concern, progression to chronic hepatitis is muchmore common after NANBH than after either Hepatitis A or Hepatitis Binfection. Chronic NANBH has been reported in 10%-70% of infectedindividuals. This form of hepatitis can be transmitted even byasymptomatic patients, and frequently progresses to malignant diseasesuch as cirrhosis and hepatocellular carcinoma. Chronic activehepatitis, with or without cirrhosis, is seen in 44%-90% ofposttransfusion hepatitis cases. Of those patients who developedcirrhosis, approximately one-fourth died of liver failure.

Chronic active NANBH is a significant problem to hemophiliacs who aredependent on blood products; 5%-11% of hemophiliacs die of chronicend-stage liver disease. Cases of NANBH other than those traceable toblood or blood products are frequently associated with hospitalexposure, accidental needle stick, or tattooing. Transmission throughclose personal contact also occurs, though this is less common for NANBHthan for Hepatitis B.

The causative agent of the majority of NANBH has been identified and isnow referred to as Hepatitis C Virus (HCV). Houghton et al., EPPublication 318,216; Choo et al., Science 1989, 244, 359-362. Based onserological studies using recombinant DNA-generated antigens it is nowclear that HCV is the causative agent of most cases of post-transfusionNANBH. The HCV genome is a positive or plus-strand RNA genome. EPPublication 318,216 (Houghton et al.) discloses partial genomicsequences of HCV-1, and teaches recombinant DNA methods of cloning andexpressing HCV sequences and HCV polypeptides, techniques of HCVimmunodiagnostics, HCV probe diagnostic techniques, anti-HCV antibodies,and methods of isolating new HCV sequences. Houghton et al. alsodisclose additional HCV sequences and teach application of thesesequences and polypeptides in immunodiagnostics, probe diagnostics,anti-HCV antibody production, PCR technology and recombinant DNAtechnology. The concept of using antisense polynucleotides as inhibitorsof viral replication is disclosed, but no specific targets are taught.oligomer probes and primers based on the sequences disclosed are alsoprovided. EP Publication 419,182 (Miyamura et al.) discloses new HCVisolates J1 and J7 and use of sequences distinct from HCV-1 sequencesfor screens and diagnostics.

The only treatment regimen shown to be effective for the treatment ofchronic NANBH is interferon-α. Most NANBH patients show an improvementof clinical symptoms during interferon treatment, but relapse isobserved in at least half of patients when treatment is interrupted.Long term remissions are achieved in only about 20% of patients evenafter 6 months of therapy. Significant improvements in antiviral therapyare therefore greatly desired. An obvious need exists for a clinicallyeffective antiviral therapy for acute and chronic HCV infections. Suchan antiviral would also be useful for preventing the development ofHCV-associated disease, for example for individuals accidently exposedto blood products containing infectious HCV. There is also a need forresearch reagents and diagnostics which are able to differentiateHCV-derived hepatitis from hepatitis caused by other agents and whichare therefore useful in designing appropriate therapeutic regimes.

Antisense Oligonucleotides

Oligonucleotides are commonly used as research reagents and diagnostics.For example, antisense oligonucleotides, which, by nature, are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes,for example to determine which viral genes are essential forreplication, or to distinguish between the functions of various membersof a biological pathway. This specific inhibitory effect has, therefore,been exploited for research use. This specificity and sensitivity isalso harnessed by those of skill in the art for diagnostic uses. Virusescapable of causing similar hepatic symptoms can be easily and readilydistinguished in patient samples, allowing proper treatment to beimplemented. Antisense oligonucleotide inhibition of viral activity invitro is useful as a means to determine a proper course of therapeutictreatment. For example, before a patient suspected of having an HCVinfection is contacted with an oligonucleotide composition of thepresent invention, cells, tissues or a bodily fluid from the patient canbe contacted with the oligonucleotide and inhibition of viral RNAfunction can be assayed. Effective in vitro inhibition of HCV RNAfunction, routinely assayable by methods such as Northern blot or RT-PCRto measure RNA replication, or Western blot or ELISA to measure proteintranslation, indicates that the infection will be responsive to theoligonucleotide treatment.

Oligonucleotides have also been employed as therapeutic moieties in thetreatment of disease states in animals and man. For example, workers inthe field have now identified antisense, triplex and otheroligonucleotide compositions which are capable of modulating expressionof genes implicated in viral, fungal and metabolic diseases. Asexamples, U.S. Pat. No. 5,166,195 issued Nov. 24, 1992, providesoligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810, issued Apr.2, 1991, provides oligomers capable of hybridizing to herpes simplexvirus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428,issued Mar. 16, 1993, provides antisense oligonucleotides havingantiviral activity against influenzavirus. U.S. Pat. No. 4,806,463,issued Feb. 21, 1989, provides antisense oligonucleotides and methodsusing them to inhibit HTLV-III replication. U.S. Pat. No. 5,276,019 andU.S. Pat. No. 5,264,423 (Cohen et al.) are directed to phosphorothioateoligonucleotide analogs used to prevent replication of foreign nucleicacids in cells. Antisense oligonucleotides have been safely andeffectively administered to humans and clinical trials of severalantisense oligonucleotide drugs are presently underway. Thephosphorothioate oligonucleotide, ISIS 2922, has been shown to beeffective against cytomegalovirus retinitis in AIDS patients. BioWorldToday, Apr. 29, 1994, p. 3. It is thus established that oligonucleotidescan be useful drugs for treatment of cells and animal subjects,especially humans.

Seki et al. have disclosed antisense compounds complementary to specificdefined regions of the HCV genome. Canadian patent application2,104,649.

Hang et al. have disclosed antisense oligonucleotides complementary tothe 5′ untranslated region of HCV for controlling translation of HCVproteins, and methods of using them. WO 94/08002.

Blum et al. have disclosed antisense oligonucleotides complementary toan RNA complementary to a portion of a hepatitis viral genome whichencodes the terminal protein region of the viral polymerase, and methodsof inhibiting replication of a hepatitis virus using sucholigonucleotides. WO 94/24864.

Wakita and Wands have used sense and antisense oligonucleotides todetermine the role of the 5′ end untranslated region in the life cycleof HCV. Antisense oligonucleotides targeted to three regions of the 5′untranslated region and one region of the core protein coding regioneffectively blocked in vitro translation of HCV protein, suggesting thatthese domains may be critical for HCV translation. J. Biol. Chem. 1994,269, 14205-14210.

SUMMARY OF THE INVENTION

In accordance with the present invention, compositions and methods formodulating the effects of HCV infection are provided. Oligonucleotideswhich are complementary to, and specifically hybridizable with, selectedsequences of HCV RNA and which are capable of inhibiting the function ofthe HCV RNA are provided. The HCV polyprotein translation initiationcodon region is a preferred target. An oligonucleotide (SEQ ID NO: 6)targeted to nucleotides 330-349 of the initiation codon region isparticularly preferred, and this sequence comprising a 5-methylcytidineat every cytidine residue is even more preferred. Methods for diagnosingor treating disease states by administering oligonucleotides, eitheralone or in combination with a pharmaceutically acceptable carrier, toanimals suspected of having HCV-associated diseases are also provided.

DETAILED DESCRIPTION OF THE INVENTION

Several regions of the HCV genome have been identified as antisensetargets in the present invention. The size of the HCV genome isapproximately 9400 nucleotides, with a single translational readingframe encoding a polyprotein which is subsequently processed to severalstructural and non-structural proteins. It should be noted that sequenceavailability and nucleotide numbering schemes vary from strain tostrain. The 5′ untranslated region (5′ UTR) or 5′ noncoding region (5′NCR) of HCV consists of approximately 341 nucleotides upstream of thepolyprotein translation initiation codon. A hairpin loop present atnucleotides 1-22 at the 5′ end of the genome (HCV-1) identified hereinas the “5′ end hairpin loop” is believed to serve as a recognitionsignal for the viral replicase or nucleocapsid proteins. Han et al.,Proc. Natl. Acad. Sci. 1991, 88, 1711-1715. The 5′ untranslated regionis believed to have a secondary structure which includes six stem-loopstructures, designated loops A-F. Loop A is present at approximatelynucleotides 13-50, loop B at approximately nucleotides 51-88, loop C atapproximately nucleotides 100-120, loop D at approximately nucleotides147-162, loop E at approximately nucleotides 163-217, and loop F atapproximately nucleotides 218-307. Tsukiyama-Kohara et al., J. Virol.1992, 66, 1476-1483. These structures are well conserved between the twomajor HCV groups.

Three small (12-16 amino acids each) open reading frames (ORFs) arelocated in the 5′-untranslated region of HCV RNA. These ORFs may beinvolved in control of translation. The ORF 3 translation initiationcodon as denominated herein is found at nucleotides 315-317 of HCV-1according to the scheme of Han et al., Proc. Natl. Acad. Sci. 1991, 88,1711-1715; and at nucleotides −127 to −125 according to the scheme ofChoo et al., Proc. Natl. Acad. Sci. 1991, 88, 2451-2455.

The polyprotein translation initiation codon as denominated herein is anAUG sequence located at nucleotides 342-344 of HCV-1 according to Han etal., Proc. Natl. Acad. Sci. 1991, 88, 1711-1715 or at nucleotide 1-3according to the HCV-1 numbering scheme of Choo et al., Proc. Natl.Acad. Sci. 1991, 88, 2451-2455. Extending downstream (toward 3′ end)from the polyprotein AUG is the core protein coding region.

The 3′ untranslated region, as denominated herein, consists ofnucleotides downstream of the polyprotein translation termination site(ending at nt 9037 according to Choo et al.; nt 9377 according toschemes of Han and Inchauspe). Nucleotides 9697-9716 (numbering schemeof Inchauspe for HCV-H) at the 3′ terminus of the genome within the 3′untranslated region can be organized into a stable hairpin loopstructure identified herein as the 3′ hairpin loop. A short nucleotidestretch (R2) immediately upstream (nt 9691-9696 of HCV-H) of the 3′hairpin, and denominated herein “the R2 sequence”, is thought to play arole in cyclization of the viral RNA, possibly in combination with a setof 5′ end 6-base-pair repeats of the same sequence at nt 23-28 and38-43. (Inchauspe et al., Proc. Natl. Acad. Sci. 1991, 88, 10292-10296)is identified herein as “5′ end 6-base-pair repeat”. Palindromesequences present near the 3′ end of the genome (nucleotides 9312-9342according to the scheme of Takamizawa et al., J. Virol. 1991, 65,1105-1113) are capable of forming a stable secondary structure. This isreferred to herein as the 3′ end palindrome region.

Antisense Oligonucleotides

The present invention employs oligonucleotides 5 to 50 nucleotides inlength which are specifically hybridizable with hepatitis C virus RNAand are capable of inhibiting the function of the HCV RNA. In preferredembodiments, oligonucleotides are targeted to the 5′ end hairpin loop,5′ end 6-base-pair repeats, 5′ end untranslated region, polyproteintranslation initiation codon, core protein coding region, ORF 3translation initiation codon, 3′-untranslated region, 3′ end palindromeregion, R2 sequence and 3′ end hairpin loop region of HCV RNA. Thisrelationship between an oligonucleotide and the nucleic acid sequence towhich it is targeted is commonly referred to as “antisense”. “Targeting”an oligonucleotide to a chosen nucleic acid target, in the context ofthis invention, is a multistep process. The process usually begins withidentifying a nucleic acid sequence whose function is to be modulated.This may be, as examples, a cellular gene (or mRNA made from the gene)whose expression is associated with a particular disease state, or aforeign nucleic acid (RNA or DNA) from an infectious agent. In thepresent invention, the target is the 5′ end hairpin loop, 5′ end6-base-pair repeats, ORF 3 translation initiation codon (all of whichare contained within the 5′ UTR), polyprotein translation initiationcodon, core protein coding region (both of which are contained withinthe coding region), 3′ end palindrome region, R2 sequence or 3′ endhairpin loop (all of which are contained within the 3′ UTR) of HCV RNA.The targeting process also includes determination of a site or siteswithin the nucleic acid sequence for the oligonucleotide interaction tooccur such that the desired effect, i.e., inhibition of HCV RNAfunction, will result. Once the target site or sites have beenidentified, oligonucleotides are chosen which are sufficientlycomplementary to the target, i.e., hybridize sufficiently well and withsufficient specificity, to give the desired modulation.

In the context of this invention “modulation” means either inhibition orstimulation. Inhibition of HCV RNA function is presently the preferredform of modulation in the present invention. The oligonucleotides areable to inhibit the function of viral RNA by interfering with itsreplication, transcription into mRNA, translation into protein,packaging into viral particles or any other activity necessary to itsoverall biological function. The failure of the RNA to perform all orpart of its function results in failure of all or a portion of thenormal life cycle of the virus. This inhibition can be measured, insamples derived from either in vitro or in vivo (animal) systems, inways which are routine in the art, for example by RT-PCR or Northernblot assay of HCV RNA levels or by in vitro translation, Western blot orELISA assay of protein expression as taught in the examples of theinstant application. “Hybridization”, in the context of this invention,means hydrogen bonding, also known as Watson-Crick base pairing, betweencomplementary bases, usually on opposite nucleic acid strands or tworegions of a nucleic acid strand. Guanine and cytosine are examples ofcomplementary bases which are known to form three hydrogen bonds betweenthem. Adenine and thymine are examples of complementary bases which formtwo hydrogen bonds between them. “Specifically hybridizable” and“complementary” are terms which are used to indicate a sufficient degreeof complementarity such that stable and specific binding occurs betweenthe DNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment or, in the case of in vitro assays,under conditions in which the assays are conducted.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of nucleotide or nucleoside monomers consistingof naturally occurring bases, sugars and intersugar (backbone) linkages.The term “oligonucleotide” also includes oligomers or polymerscomprising non-naturally occurring monomers, or portions thereof, whichfunction similarly. Such modified or substituted oligonucleotides areoften preferred over native forms because of properties such as, forexample, enhanced cellular uptake, increased stability in the presenceof nucleases, or enhanced target affinity. A number of nucleotide andnucleoside modifications have been shown to make the oligonucleotideinto which they are incorporated more resistant to nuclease digestionthan the native oligodeoxynucleotide. Nuclease resistance is routinelymeasured by incubating oligonucleotides with cellular extracts orisolated nuclease solutions and measuring the extent of intactoligonucleotide remaining over time, usually by gel electrophoresis.Oligonucleotides which have been modified to enhance their nucleaseresistance survive intact for a longer time than unmodifiedoligonucleotides. A number of modifications have also been shown toincrease binding (affinity) of the oligonucleotide to its target.Affinity of an oligonucleotide for its target is routinely determined bymeasuring the Tm of an oligonucleotide/target pair, which is thetemperature at which the oligonucleotide and target dissociate.Dissociation is detected spectrophotometrically. The higher the Tm, thegreater the affinity of the oligonucleotide for the target. In somecases, oligonucleotide modifications which enhance target bindingaffinity are also, independently, able to enhance nuclease resistance.

Specific examples of some preferred oligonucleotides envisioned for thisinvention may contain phosphorothioates (P=S), phosphotriesters, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages orshort chain heteroatomic or heterocyclic intersugar (“backbone”)linkages at one or more positions instead of the native phosphodiester(P=O) backbone. Most preferred are phosphorothioates and those withCH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂ [known as a methylene(methylimino) or MMIbackbone], CH₂—O—N(CH₃) —CH₂, CH₂—N(CH₃) —N(CH₃) —CH₂ andO—N(CH₃)—CH₂—CH₂ backbones (where phosphodiester is O'P—O—CH₂). Alsopreferred are oligonucleotides having morpholino backbone structures.Summerton, J. E. and Weller, D. D., U.S. Pat. No. 5,034,506. In otherpreferred embodiments, such as the protein-nucleic acid orpeptide-nucleic acid (PNA) backbone, the phosphodiester backbone of theoligonucleotide may be replaced with a polyamide backbone, the basesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone. P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt,Science 1991, 254, 1497. Oligonucleotides containing one or more PNA,MMI or P=S backbone linkages are presently more preferred. Otherpreferred oligonucleotides may contain one or more substituted sugarmoieties comprising one of the following at the 2′ position: OH, SH,SCH₃, F, OCN, OCH₃OCH₃, OCH₃O(CH₂)_(n)CH₃, O(CH₂)_(n)NH₂ orO(CH₂)_(n)CH₃ where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN;CF₃; OCF₃; O—, S—, or N—alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleavinggroup; a cholesteryl group; a reporter group; an intercalator; a groupfor improving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. Presently preferredmodifications include 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃), 2′-methoxy(2′—O—CH₃), 2′—propoxy (2′—OCH₂CH₂CH₃) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutyls inplace of the pentofuranosyl group.

The oligonucleotides of the invention may additionally or alternativelyinclude nucleobase modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include adenine (A), guanine (G),thymine (T), cytosine (C) and uracil (U). Modified nucleobases known inthe art include nucleobases found only infrequently or transiently innatural nucleic acids, e.g., hypoxanthine (whose correspondingnucleotide, inosine, is sometimes referred to as a “universal base”) ,6-methyladenine, 5-methylcytosine, 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentiobiosyl HMC, as well synthetic nucleobases, e.g.,5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine,N⁶(6-aminohexyl)adenine and 2,6-diaminopurine. Oligonucleotides in whichcytosine bases are replaced by 5-methylcytosines are presently apreferred embodiment of the invention.

Another preferred additional or alternative modification of theoligonucleotides of the invention involves chemically linking to theoligonucleotide one or more lipophilic moieties which enhance thecellular uptake of the oligonucleotide. Such lipophilic moieties may belinked to an oligonucleotide at several different positions on theoligonucleotide. Some preferred positions include the 3′ position of thesugar of the 3′ terminal nucleotide, the 5′ position of the sugar of the5′ terminal nucleotide, and the 2′ position of the sugar of anynucleotide. The N⁶ position of a purine nucleobase may also be utilizedto link a lipophilic moiety to an oligonucleotide of the invention. Suchlipophilic moieties known in the art include but are not limited to oneor more cholesteryl moieties, cholic acids, thioethers,thiocholesterols, aliphatic chains, e.g., dodecandiol or undecylresidues, phospholipids, polyamines or polyethylene glycol chains,adamantane acetic acid, palmityl moieties, octadecylamine orhexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides comprisinglipophilic moieties, and methods for preparing such oligonucleotides, asdisclosed in U.S. Pat. Nos. 5,138,045, No. 5,218,105 and No. 5,459,255,the contents of which are hereby incorporated by reference.

Certain preferred oligonucleotides of this invention are chimericoligonucleotides. “Chimeric oligonucleotides” or “chimeras”, in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionof modified nucleotides that confers one or more beneficial properties(such as, for example, increased nuclease resistance, increased uptakeinto cells, increased binding affinity for the RNA target) and a regionthat is a substrate for RNase H cleavage. In one preferred embodiment, achimeric oligonucleotide comprises at least one region modified toincrease target binding affinity, and, usually, a region that acts as asubstrate for RNAse H. Affinity of an oligonucleotide for its target (inthis case a nucleic acid encoding HCV RNA) is routinely determined bymeasuring the Tm of an oligonucleotide/target pair, which is thetemperature at which the oligonucleotide and target dissociate;dissociation is detected spectrophotometrically. The higher the Tm, thegreater the affinity of the oligonucleotide for the target. In a morepreferred embodiment, the region of the oligonucleotide which ismodified to increase HCV RNA binding affinity comprises at least onenucleotide modified at the 2′ position of the sugar, most preferably a2′-O-alkyl or 2′-fluoro-modified nucleotide. Such modifications areroutinely incorporated into oligonucleotides and these oligonucleotideshave been shown to have a higher Tm (i.e., higher target bindingaffinity) than 2′-deoxyoligonucleotides against a given target. Theeffect of such increased affinity is to greatly enhance antisenseoligonucleotide inhibition of HCV RNA function. RNAse H is a cellularendonuclease that cleaves the RNA strand of RNA:DNA duplexes; activationof this enzyme therefore results in cleavage of the RNA target, and thuscan greatly enhance the efficiency of antisense inhibition. Cleavage ofthe RNA target can be routinely demonstrated by gel electrophoresis. Inanother preferred embodiment, the chimeric oligonucleotide is alsomodified to enhance nuclease resistance. Cells contain a variety of exo-and endo-nucleases which can degrade nucleic acids. A number ofnucleotide and nucleoside modifications have been shown to make theoligonucleotide into which they are incorporated more resistant tonuclease digestion than the native oligodeoxynucleotide. Nucleaseresistance is routinely measured by incubating oligonucleotides withcellular extracts or isolated nuclease solutions and measuring theextent of intact oligonucleotide remaining over time, usually by gelelectrophoresis. oligonucleotides which have been modified to enhancetheir nuclease resistance survive intact for a longer time thanunmodified oligonucleotides. A variety of oligonucleotide modificationshave been demonstrated to enhance or confer nuclease resistance. In somecases, oligonucleotide modifications which enhance target bindingaffinity are also, independently, able to enhance nuclease resistance.oligonucleotides which contain at least one phosphorothioatemodification are presently more preferred.

The compounds of the present invention include bioequivalent compounds,including pharmaceutically acceptable salts and prodrugs.

The compounds of the invention encompass any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto pharmaceutically acceptable salts of the nucleic acids of theinvention and prodrugs of such nucleic acids.

Pharmaceutically acceptable salts are physiologically andpharmaceutically acceptable salts of the nucleic acids of the invention,i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci. 1977, 66:1). The base addition salts of said acidic compounds areprepared by contacting the free acid form with a sufficient amount ofthe desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid or phosphoric acid; with organiccarboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamicacids, for example acetic acid, propionic acid, glycolic acid, succinicacid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid,malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid,glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, salicylic acid, 4-aminosalicylic acid,2-phenoxybenzoic acid, 2-acetoxybenzoic acid, nicotinic acid orisonicotinic acid; and with amino acids, such as the 20 alpha-aminoacids involved in the synthesis of proteins in nature, for exampleglutamic acid or aspartic acid, and also with phenylacetic acid,methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid,ethane-1,2-disulfonic acid, benzenesulfonic acid,4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid,naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate,glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation ofcyclamates), or with other acid organic compounds, such as ascorbicacid.

Pharmaceutically acceptable salts of compounds may also be formed with apharmaceutically acceptable cation. Suitable pharmaceutically acceptablecations are well known to those skilled in the art and include alkaline,alkaline earth, ammonium and quaternary ammonium cations. Carbonates orhydrogen carbonates are also possible.

For oligonucleotides, examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as acetic acid, oxalic acid, tartaricacid, succinic acid, maleic acid, fumaric acid, gluconic acid, citricacid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmiticacid, alginic acid, polyglutamic acid, naphthalenesulfonic acid,methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonicacid, polygalacturonic acid, and the like; and (d) salts formed fromelemental anions such as chlorine, bromine, and iodine.

The oligonucleotides of the invention may additionally or alternativelybe prepared to be delivered in a prodrug form. The term “prodrug”indicates a therapeutic agent that is prepared in an inactive form thatis converted to an active form (i.e., drug) within the body or cellsthereof by the action of endogenous enzymes or other chemicals and/orconditions. In particular, prodrug versions of the oligonucleotides ofthe invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate]derivatives according to the methods disclosed in WO 93/24510 toGosselin et al., published Dec. 9, 1993.

The oligonucleotides in accordance with this invention preferably arefrom about 5 to about 50 nucleotides in length. In the context of thisinvention it is understood that this encompasses non-naturally occurringoligomers as hereinbefore described, having 5 to 50 monomers.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is also well known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. It is also well known touse similar techniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as those available from GlenResearch, Sterling Va., to synthe-size modified oligonucleotides such ascholesterol-modified oligonucleotides.

Methods of modulating the activity of HCV virus are provided, in whichthe virus, or cells, tissues or bodily fluid suspected of containing thevirus, is contacted with an oligonucleotide of the invention. In thecontext of this invention, to “contact” means to add the oligonucleotideto a preparation of the virus, or vice versa, or to add theoligonucleotide to a preparation or isolate of cells, tissues or bodilyfluid, or vice versa, or to add the oligonucleotide to virus, cellstissues or bodily fluid in situ, i.e., in an animal, especially a human.

The oligonucleotides of this invention can be used in diagnostics,therapeutics and as research reagents and kits. Since theoligonucleotides of this invention hybridize to RNA from HCV, sandwichand other assays can easily be constructed to exploit this fact.Provision of means for detecting hybridization of oligonucleotide withHCV or HCV RNA present in a sample suspected of containing it canroutinely be accomplished. Such provision may include enzymeconjugation, radiolabelling or any other suitable detection systems.Kits for detecting the presence or absence of HCV may also be prepared.The specific ability of the oligonucleotides of the invention to inhibitHCV RNA function can also be exploited in the detection and diagnosis ofHCV, HCV infection and HCV-associated diseases. As described in theexamples of the present application, the decrease in HCV RNA or proteinlevels as a result of oligonucleotide inhibition of HCV RNA function canbe routinely detected, for example by RT-PCR, Northern blot, Westernblot or ELISA.

For prophylactics and therapeutics, methods of preventing HCV-associateddisease and of treating HCV infection and HCV-associated disease areprovided. The formulation of therapeutic compositions and theirsubsequent administration is believed to be within the skill in the art.Oligonucleotides may be formulated in a pharmaceutical composition,which may include carriers, thickeners, diluents, buffers,preservatives, surface active agents, liposomes or lipid formulationsand the like in addition to the oligonucleotide. Pharmaceuticalcompositions may also include one or more active ingredients such asinterferons, antimicrobial agents, anti-inflammatory agents,anesthetics, and the like. Formulations for parenteral administrationmay include sterile aqueous solutions which may also contain buffers,liposomes, diluents and other suitable additives.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous drip, subcutaneous, intraperitonealor intramuscular injection, pulmonary administration, e.g., byinhalation or insufflation, or intracranial, e.g., intrathecal orintraventricular, administration. For oral administration, it has beenfound that oligonucleotides with at least one 2′-substitutedribonucleotide are particularly useful because of their absorption anddistribution characteristics. U.S. Pat. No. 5,591,721 issued to Agrawalet al. Oligonucleotides with at least one 2′-O-methoxyethyl modificationare believed to be particularly useful for oral administration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

Dosing is dependent on severity and responsiveness of the condition tobe treated, with course of treatment lasting from several days toseveral months or until a reduction in viral titer (routinely measuredby Western blot, ELISA, RT-PCR, or RNA (Northern) blot, for example) iseffected or a diminution of disease state is achieved. Optimal dosingschedules are easily calculated from measurements of drug accumulationin the body. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Therapeutically orprophylactically effective amounts (dosages) may vary depending on therelative potency of individual compositions, and can generally beroutinely calculated based on molecular weight and EC50s in in vitroand/or animal studies. For example, given the molecular weight of drugcompound (derived from oligonucleotide sequence and chemical structure)and an experimentally derived effective dose such as an IC₅₀, forexample, a dose in mg/kg is routinely calculated. In general, dosage isfrom 0.001 μg to 100 g and may be administered once or several timesdaily, weekly, monthly or yearly, or even every 2 to 20 years.

Pharmacokinetics of Antisense Oligonucleotides

Because the primary pathology associated with HCV infection occurs inthe liver of infected individuals, the ability of a potential anti-HCVcompound to achieve significant concentrations in the liver isadvantageous. Pharmacokinetic profiles for a number of oligonucleotides,primarily phosphorothioate oligonucleotides, have been determined.Phosphorothioate oligonucleotides have been shown to have very similarpharmacokinetics and tissue distribution, regardless of sequence. Thisis characterized in plasma by a rapid distribution phase (approximately30 minutes) and a prolonged elimination phase (approximately 40 hours).Phosphorothioates are found to be broadly distributed to peripheraltissues (i.e., excepting the brain, which is reachable directly, e.g.,by intraventricular drug administration, and in addition may bereachable via a compromised blood-brain barrier in many nervous systemconditions), with the highest concentrations found in liver, renalcortex and bone marrow. There is good accumulation of intact compound inmost tissues, particularly liver, kidney and bone marrow, with veryextended compound half-life in tissues. Similar distribution profilesare found whether the oligonucleotide is administered intravenously orsubcutaneously. Furthermore, the pharmacokinetic and tissue distributionprofiles are very consistent among animal species, including rodents,monkeys and humans.

Preferred Embodiments of the Invention

It has been found that antisense oligonucleotides designed to targetviruses can be effective in diminishing viral infection.

In accordance with this invention, persons of ordinary skill in the artwill understand that messenger RNA includes not only the sequenceinformation to encode a protein using the three letter genetic code, butalso associated ribonucleotides which form regions known to such personsas the 5′-untranslated region, the 3′-untranslated region, and the 5′cap region, as well as ribonucleotides which form various secondarystructures. Thus, oligonucleotides may be formulated in accordance withthis invention which are targeted wholly or in part to these associatedribonucleotides as well as to the coding ribonucleotides. In preferredembodiments, the oligonucleotide is specifically hybridizable with theHCV 5′ end hairpin loop, 5′ end 6-base-pair repeats, ORF 3 translationinitiation codon, (all of which are contained within the 5′ UTR)polyprotein translation initiation codon, core protein coding region(both of which are contained within the coding region) , R2 region, 3′hairpin loop or 3′ end palindrome region (all of which are containedwithin the 3′-untranslated region).

It is to be expected that differences in the RNA of HCV from differentstrains and from different types within a strain exist. It is believedthat the regions of the various HCV strains serve essentially the samefunction for the respective strains and that interference withhomologous or analogous RNA regions will afford similar results in thevarious strains. This is believed to be so even though differences inthe nucleotide sequences among the strains exist.

Accordingly, nucleotide sequences set forth in the present specificationwill be understood to be representational for the particular strainbeing described. Homologous or analogous sequences for different strainsof HCV are specifically contemplated as being within the scope of thisinvention. In preferred embodiments of the present invention, antisenseoligonucleotides are targeted to the 5′ untranslated region, coreprotein translation initiation codon region, core protein coding region,ORF 3 translation initiation codon and 3′-untranslated region of HCVRNA.

In preferred embodiments, the antisense oligonucleotides arehybridizable with at least a portion of the polyprotein translationinitiation codon or with at least a portion of the core protein codingregion. The sequence of nucleotides 1-686 (SEQ ID NO: 37) comprises theentire 5′-untranslated region (nucleotides 1-341) and a 145-nucleotidecore region sequence of HCV RNA. A highly preferred oligonucleotidehybridizable with at least a portion of the polyprotein translationinitiation codon comprises SEQ ID NO: 6.

In vitro Evaluation of HCV Antisense Oligonucleotides

HCV replication in cell culture has not yet been achieved. Consequently,in vitro translation assays are used to evaluate antisenseoligonucleotides for anti-HCV activity. One such in vitro translationassay was used to evaluate oligonucleotide compounds for the ability toinhibit synthesis of HCV 5′ UTR-core-env transcript in a rabbitreticulocyte assay.

Cell-based assays are also used for evaluation of oligonucleotides foranti-HCV activity. In one such assay, effects of oligonucleotides on HCVRNA function are evaluated by measuring RNA and/or HCV core proteinlevels in transformed hepatocytes expressing the 5′end of the HCVgenome. Recombinant HCV/vaccinia virus assays can also be used, such asthose described in the examples of the present application. Luciferaseassays can be used, for example, as described in the examples of thepresent application, in which recombinant vaccinia virus containing HCVsequences fused to luciferase sequences are used. Quantitation ofluciferase with a luminometer is a simple way of measuring HCV coreprotein expression and its inhibition by antisense compounds. This canbe done in cultured hepatocytes or in tissue samples, such as liverbiopsies, from treated animals.

Animal Models for HCV

There is no small animal model for chronic HCV infection. A recombinantvaccinia/HCV/luciferase virus expression assay has been developed fortesting compounds in mice. Mice are inoculated with recombinant vacciniavirus (either expressing HCV/luciferase or luciferase alone for acontrol). Organs (particularly liver) are harvested one or more dayslater and luciferase activity in the tissue is assayed by luminometry.

The following specific examples are provided for illustrative purposesonly and are not intended to limit the invention.

EXAMPLES Example 1 Oligonucleotide Synthesis

Unmodified oligodeoxynucleotides were synthesized on an automated DNAsynthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl-phosphoramidites were purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of³H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step.

2′-methoxy oligonucleotides were synthesized using 2′-methoxyβ-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham Mass.) andthe standard cycle for unmodified oligonucleotides, except the wait stepafter pulse delivery of tetrazole and base was increased to 360 seconds.Other 2′-alkoxy oligonucleotides were synthesized by a modification ofthis method, using appropriate 2′-modified amidites such as thoseavailable from Glen Research, Inc., Sterling, Va.

2′-fluoro oligonucleotides were synthesized as described in Kawasaki etal., J. Med. Chem. 1993, 36, 831. Briefly, the protectednucleosideN⁶-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizingcommercially available 9-8-D-arabinofuranosyladenine as startingmaterial and by modifying literature procedures whereby the 2′-″-fluoroatom is introduced by a S_(N)2-displacement of a 2′-8-O-trifyl group.Thus N⁶-benzoyl-9-8-D-arabinofuranosyladenine was selectively protectedin moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N⁶-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected 9-8-D-arabinofuranosylguanineas starting material, and conversion to the intermediatediisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS groupwas followed by protection of the hydroxyl group with THP to givediisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which 2,2′-anhydro-1-8-D-arabinofuranosyluracil was treated with 70% hydrogenfluoride-pyridine. Standard procedures were used to obtain the 5′-DMTand 5′-DMT-3′ phosphoramidites.

2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN⁴-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

Oligonucleotides having methylene(methylimino) backbones are synthesizedaccording to U.S. Pat. No. 5,378,825, which is coassigned to theassignee of the present invention and is incorporated herein in itsentirety. Other nitrogen-containing backbones are synthesized accordingto WO 92/20823 which is also coassigned to the assignee of the presentinvention and incorporated herein in its entirety.

Oligonucleotides having amide backbones are synthesized according to DeMesmaeker et al., Acc. Chem. Res. 1995, 28, 366. The amide moiety isreadily accessible by simple and well-known synthetic methods and iscompatible with the conditions required for solid phase synthesis ofoligonucleotides.

Oligonucleotides with morpholino backbones are synthesized according toU.S. Pat. No. 5,034,506 (Summerton and Weller).

Peptide-nucleic acid (PNA) oligomers are synthesized according to P. E.Nielsen et al., Science 1991, 254, 1497).

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55ECfor 18 hours, the oligonucleotides are purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotideswere analyzed by polyacrylamide gel electrophoresis on denaturing gelsand judged to be at least 85% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in synthesiswere periodically checked by ³¹P nuclear magnetic resonancespectroscopy, and for some studies oligonucleotides were purified byHPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162.Results obtained with HPLC-purified material were similar to thoseobtained with non-HPLC purified material.

Oligonucleotides having 2′—O—CH₂CH₂OCH₃ modified nucleotides weresynthesized according to the method of Martin. Helv. Chim. Acta 1995,78, 486-504. All 2′—O—CH₂CH₂OCH₃ cytosines were 5-methyl cytosines,synthesized as follows:

Monomers 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid which was crushed to a light tan powder (57 g, 85%crude yield). The material was used as is for further reactions.

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL) The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57w).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl_(3.) The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tlc showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHC₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

5-methylcytidine DMT β-cyanoethyl phosphoramidites are commerciallyavailable from PerSeptive Biosystems (Framingham, Mass.).

Example 2 Evaluation of Inhibitory Activity of Antisenseoligonucleotides which are targeted to the polyprotein translationinitiation codon region and adjacent core protein coding region

(1) In order to evaluate the inhibitory activity of antisenseoligonucleotides which are complementary to the region including thetranslation initiation codon (nucleotide number 342-344) of HCV-RNA andthe adjacent core protein coding region, a series of 20 mer antisenseoligonucleotides were prepared which are complementary to the regionfrom nucleotide 320 to nucleotide 379. These are named according totheir target sequence on the HCV RNA, i.e., the oligonucleotide name(e.g., 330) is the number of the 5′-most nucleotide of the correspondingHCV RNA target sequence shown in SEQ ID NO: 37. Accordingly,oligonucleotide 330 is targeted to nucleotides 330-349 of the HCV RNAshown in SEQ ID NO: 37. Of these oligonucleotides, oligonucleotides 324through 344 contain all or part of the sequence CAT which iscomplementary to the AUG initiation codon itself. The nucleotidesequence of these antisense oligonucleotides are shown in Table 1.

TABLE 1 Antisense oligonucleotides to HCV SEQ ID Oligo Sequence %Inhibition NO: 320 TGC ACG GTC TAC GAG ACC TC  3  1 322 GGT GCA CGG TCTACG AGA CC  5  2 324 ATG GTG CAC GGT CTA CGA GA 31  3 326 TCA TGG TGCACG GTC TAC GA 39  4 328 GCT CAT GGT GCA CGG TCT AC 71  5 330 GTG CTCATG GTG CAC GGT CT 38  6 332 TCG TGC TCA TGG TGC ACG GT  5  7 334 ATTCGT GCT CAT GGT GCA CG 39  8 336 GGA TTC GTG CTC ATG GTG CA 98  9 338TAG GAT TCG TGC TCA TGG TG 99 10 340 TTT AGG ATT CGT GCT CAT GG 97 11342 GGT TTA GGA TTC GTG CTC AT 96 12 344 GAG GTT TAG GAT TCG TGC TC 9913 344-i1 GAG GTT TAG GAT TIG TGC TC 95 14 344-i3 GIG GTT TIG GAT TIGTGC TC 90 15 344-i5 GIG GTT TIG GAI JIG TGC TC 51 16 346 TTG AGG TTT AGGATT CGT GC 98 17 348 CTT TGA GGT TTA GGA TTC GT 98 18 350 TTC TTT GAGGTT TAG GAT TC 99 19 352 TTT TCT TTG AGG TTT AGG AT 99 20 354 GTT TTTCTT TGA GGT TTA GG 91 21 356 TGG TTT TTC TTT GAG GTT TA 86 22 358 TTTGGT TTT TCT TTG AGG TT 83 23 360 CGT TTG GTT TTT CTT TGA GG 81 24

The inhibitory activity of these 21 antisense oligonucleotides wasevaluated in the in vitro translation assay. As shown in Table 1,antisense oligonucleotides 328, 336, 338, 340, 342, 344, 346, 348, 350,352, 354, 356, 358 and 360 showed an inhibitory activity of greater than70%, and are preferred. Of these, 336, 338, 340, 342, 344, 346, 348, 350and 352 showed an extremely high inhibitory activity of over 95% and aremost preferred.

The HCV target sequence regions complementary to the above 9 most activeantisense oligonucleotides have in common the four nucleotides fromnumber 352 to 355 in the core protein coding region near the polyproteintranslation initiation codon. Thus, it is preferred to target these fournucleotides in order to inhibit the translation. Accordingly,oligonucleotides comprising the sequence GGAT are preferred embodimentsof the invention.

(2) Evaluation of antisense oligonucleotides in which the nucleotidesknown to be variable among strains were replaced by inosine:

It is known that in the nucleotide sequences in the core protein codingregion near the translation initiation codon, variation of bases amongstrains occasionally occurs at nucleotides 350, 351, 352, 356 and 362.Based on this knowledge, it was studied whether substitution of thesebases by the “universal base” inosine would be effective for inhibitionof various viruses.

An antisense DNA, designated oligonucleotide 344-il, was prepared inwhich the base at base number 350 in oligonucleotide 344 was replaced byinosine. Likewise, an antisense DNA, designated oligonucleotide 344-i3,in which three bases at base numbers 350, 356 and 362 were substitutedby inosine, and an antisense DNA, designated oligonucleotide 344-i5, inwhich five bases at base numbers 350, 351, 352, 356, and 362 weresubstituted by inosine, were prepared. The inhibitory activity of theseantisense oligonucleotides was evaluated in the in vitro translationassay. As a result, oligonucleotides 344-il and 344-i3 showed highinhibitory activity. Therefore, antisense oligonucleotides targeted tonucleotides 344-363 of HCV RNA and which have three inosine substituentsor less are preferred. Their inhibitory activities are shown in Table 1.

Example 3 Evaluation of Oligonucleotides 120, 330 and 340 and truncatedversions of oligonucleotides 120, 260, 330 and 340 in H8Adl7 cell assayfor effects on HCV RNA levels

The anti-HCV activity of P=S oligonucleotides 120, 330 and 340 wasevaluated in H8Adl7 cells as follows.

An expression plasmid containing a gene (1.3 kb) coding for 5′NCR-core-env region of HCV gene was prepared by conventional methods andtransfected into a liver cell strain (H8Adl7) by lipofection accordingto standard methods. The desired liver cell transformant, whichexpressed HCV core protein, was obtained.

HCV RNA was isolated and quantitated by Northern blot analysis todetermine levels of expression. Core protein expression could also bedetected by ELISA method using an anti-HCV core-mouse monoclonalantibody as the solid phase antibody; an anti-HCV human polyclonalantibody as the primary antibody; and an HRP (horseradish peroxidase)-conjugated anti-human IgG-mouse monoclonal antibody as the secondaryantibody.

The liver cell transformant (2.5×10⁵ cells) were inoculated on 6-wellplates. To each plate was added each of the above-obtained fiveantisense oligonucleotides (each at a concentration of 5 μM). After twodays, the cells were harvested and counted. The cells were washed onceand lysed, and the inhibitory activity was measured by Northern blot.The inhibitory activities of the P=S antisense oligonucleotides werecalculated, compared to control without antisense oligonucleotide.

As before, the oligonucleotide number is the number of the 5′-mostnucleotide of the corresponding HCV RNA target sequence shown in SEQ IDNO: 37. For example, oligonucleotide 120 is a 20 mer targeted tonucleotides 120-139 of HCV RNA. Each of these compounds inducedreduction in HCV RNA levels at doses of 0.5 μM and 0.17 μM. These threecompounds (P=S 20 mers 120, 330 and 340) are therefore highly preferred.15 mer versions (truncated at by 5 nucleotides at either the 3′ or 5′end) induced a reduction of HCV RNA at the 0.5 μM dose. These compoundsare therefore preferred. 10 mers did not show sequence-specificinhibition at either dose.

A number of shortened analogs of oligonucleotide 330 were alsosynthesized as phosphorothioates and evaluated for effects on HCV RNAlevels in the same manner. The sequence of oligonucleotide 330 wastruncated at one or both ends. These oligonucleotides are shown in Table2. Oligonucleotide concentration was 100 nM.

TABLE 2 Activity % SEQ ID Oligo Sequence control NO 330 GTG CTC ATG GTGCAC GGT CT 30%  6 9559 GTG CTC ATG GTG CAC GGT 53 25 9557 GTG CTC ATGGTG CAC GG 52 26 9558 GTG CTC ATG GTG CAC G 66 27 9036 GTG CTC ATG GTGCAC 37 28 9035 GTG CTC ATG G 100 29 10471   G CTC ATG GTG CAC GGT CT 2730 10470     CTC ATG GTG CAC GGT CT 35 31 9038       C ATG GTG CAC GGTCT 32 32 9034              TG CAC GGT CT 82 33 10549  TG CTC ATG GTG CACGGT C 17 34 10550   G CTC ATG GTG CAC GGT 36 35

In this assay, oligonucleotides 9036, 10471, 10470, 9038, 10549 and10550 gave greater than 50% inhibition of HCV RNA ion and are thereforepreferred.

Example 4

Evaluation of oligos 259, 260 and 330 in the HCV H8Adl7 RNA assay

The anti-HCV activity of P=S and 2′-O-propyl/P=S gapped oligonucleotideswas evaluated in H8Ad17 cells as described in Example 3. P=Soligonucleotides 259, 260 and 330 all induced similar (approx 55%)reduction in HCV RNA levels in this assay, using 70 nM oligonucleotideconcentration. The 2′-O-propyl gapped version of oligonucleotide 259showed approximately 25% inhibition of HCV RNA levels (170 nM oligodose), but oligonucleotides 260 and 330 were not active as 2′-O-propylgapped oligonucleotides in this assay. In a previous assay of the sametype, the gapped 2′-O-propyl version of oligonucleotide 330 did induce areduction of HCV RNA, though less than was observed for the P=S 330oligonucleotide.

Example 5 Evaluation of oligos 259, 260 and 330 in an HCV H8Adl7 proteinassay

A Western blot assay employing affinity-purified human polyclonalanti-HCV serum and ¹²⁵I-conjugated goat anti-human IgG was developed inplace of ELISA assays previously used to evaluate effects ofoligonucleotides on HCV core protein levels. Six-well plates were seededwith H8 cells at 3.5×10⁵ cells/well. Cells were grown overnight. Cellswere treated with oligonucleotide in Optimem containing 5 μg/mllipofectin for 4 hours. Cells were fed with 2 ml H8 medium and allowedto recover overnight. To harvest cells, cells were washed once with 2 mlPBS, lysed in 100 μl Laemmli buffer and harvested by scraping. Forelectrophoresis, cell lysates were boiled, and 10-14 Al of cell lysatewas loaded on each lane of a 16% polyacrylamide gel. Afterelectrophoresing, proteins were transferred electrophoretically ontoPVDF membrane. The membrane was blocked in PBS containing 2% goat serumand 0.3% TWEEN-20, and incubated overnight with primary antibody (humananti-core antibody 2243 and rabbit anti-G3PDH antibody). The membranewas washed 5×5 minutes in buffer, then incubated with secondaryantibodies for 4-8 hours (¹²⁵I-conjugated goat anti-human, and¹²⁵I-conjugated goat anti-rabbit) . The membrane was washed 5×5 minutesin buffer, sealed in plastic and exposed in a PhosphorImager cassetteovernight. Bands were quantitated on the PhosphorImager (MolecularDynamics, Sunnyvale Calif.), normalized to G3PDH expression levels, andresults were plotted as a percentage of control untreated cells.

P=S and 2′-modified 330 oligonucleotides were evaluated using thisWestern blot assay. These oligonucleotides are shown in Table 3. In thesequences shown, capital letters represent base sequence, small letters(o or s) represent internucleoside linkage, either phosphodiester (P=O)or phosphorothioate (P=S), respectively. Bold=2′-O-propyl.*=2′-O-butylimidazole. +=2′-O-propylamine.

TABLE 3 SEQ ID Oligo # Sequence NO 330GsTsGsCsTsCsAsTsGsGsTsGsCsAsCsGsGsTsCsT 6 330GsTsGsCsTsCsAsTsGsGsTsGsCsAsCsGsGsTsCsT 6* *                               * * 330GsTsGsCsTsCsAsTsGsGsTsGsCsAsCsGsGsTsCsT 6+ +                               + + 330GsTsGsCsTsCsAsTsGsGsTsGsCsAsCsGsGsTsCsT 6

Cells were treated with oligonucleotide at doses of 25 nM, 100 nM or 400nM. The greatest reduction in core protein (approx 90-95% at higherdoses) was observed with the P=S oligonucleotide. This compound istherefore highly preferred. The 2′-O-propyl gapped P=S oligonucleotidegave approximately 80% inhibition of core protein expression. Thiscompound is therefore preferred. The 2′-O-propyl/P=O compound did notshow activity in this assay.

Example 6 Evaluation of modified 330 oligos in cellular assays

Oligonucleotides with the 330 sequence (SEQ ID NO: 6) and containingvarious modifications [P=S deoxy; 2′-O-propyl (uniform 2′-O-propyl or2′-O-propyl gapped, both uniformly P=S); or 2′-fluoro modifications(gapped or uniform, both uniformly P=S)] were evaluated in the H8Adl7core protein Western blot assay compared to a scrambled phosphorothioatecontrol.

In this assay, the P=S oligonucleotide was consistently the best, givingan average of 62.4% inhibition of HCV core protein translation (n=7)compared to control. 2′-O-propyl and 2′-fluoro gapped oligonucleotidesgave over 50% inhibition in at least one experiment. Uniformly 2′-fluoroor uniformly 2′-O-propyl oligonucleotides were inactive in thisexperiment.

In this assay, the P=S oligonucleotides were consistently the best andare preferred. Of these, P=S oligonucleotides 260, 270, 275, 277 and 330are more preferred. Uniform 2′fluoro P=S oligonucleotides 345, 347 and355 are also more preferred.

Additional uniform 2′-fluoro phosphorothioate oligonucleotides weresynthesized and tested for ability to inhibit HCV core proteinexpression. oligonucleotide 344 was also found to be extremely activeand is preferred. The region of the HCV RNA target from nucleotide 344to nucleotide 374 was found to be extremely sensitive to antisenseoligonucleotide inhibition. Oligonucleotides complementary to thistarget region, therefore, are preferred. More preferred among these arethe 2′fluoro phosphorothioate oligonucleotides.

Example 7 Evaluation of a “single virus” recombinant vaccinia/HCV coreprotein assay

A “single virus” vaccinia assay system was developed, which does notrequire co-infection with helper vaccinia virus expressing T7polymerase. Cells were pretreated with oligonucleotide in the absence oflipofectin prior to infection with recombinant vaccinia virus expressingHCV sequences. Cells were then infected with recombinant vaccinia virusexpressing HCV 5′ UTR-core at a m.o.i. of 2.0 pfu/cell. After infection,cells were rinsed and post-treated with medium containingoligonucleotide. Initial results obtained with this assay indicate thatP=S oligonucleotides 259 and 260 inhibit HCV 5′-UTR core expressionby >60% at a concentration of 1 μM. Inhibition is dose-dependent.

Uniformly 2′-fluoro P=S oligonucleotides 260, 330 and 340 were evaluatedfor activity in the recombinant vaccinia “single virus” assay using RY5cells. Medium containing oligonucleotide was added after infection.2′-fluoro modified oligonucleotide 260 induced a dose-dependentinhibitory effect on HCV core protein expression (up to approximately65% inhibition) even without pretreatment of cells with oligonucleotidebefore infection. In the same assay with pretreatment, 2′-fluoro P=Smodified oligonucleotide 340 effectively inhibited HCV core proteinexpression at doses of 0.1 μM, 0.3 μM and 1.0 μM, with a maximuminhibition of about 75%. This oligonucleotide is therefore preferred. Inthe “single virus” assay using HepG2 cells, a dose-dependent inhibitoryeffect of oligonucleotide 340 as a uniform 2′-fluoro phosphorothioatewas also observed (approximately 60% inhibition). This oligonucleotideis therefore preferred. The phosphorothioate oligonucleotide 260 alsogave approximately 60% inhibition in the HepG2 cell assay.

Example 8 Diagnostic use of Oligonucleotides which inhibit HCV

Definitive diagnosis of HCV-caused hepatitis can be readily accomplishedusing antisense oligonucleotides which inhibit HCV RNA function,measurable as a decrease in HCV RNA levels or HCV core protein levels.RNA is extracted from blood samples or liver tissue samples obtained byneedle biopsy, and electrophoresed and transferred to nitrocellulose forNorthern blotting according to standard methods routinely used by thoseskilled in the art. An identical sample of blood or tissue is treatedwith antisense oligonucleotide prior to RNA extraction. The intensity ofputative HCV signal in the two blots is then compared. If HCV is present(and presumably causative of disease), the HCV RNA signal will bereduced in the oligonucleotide-treated sample compared to the untreatedsample. If HCV is not the cause of the disease, the two samples willhave identical signals. Similar assays can be designed which employother methods such as RT-PCR for HCV RNA detection and quantitation, orWestern blotting or ELISA measurement of HCV core protein translation,all of which are routinely performed by those in the art.

Diagnostic methods using antisense oligonucleotides capable ofinhibiting HCV RNA function are also useful for determining whether agiven virus isolated from a patient with hepatitis will respond totreatment, before such treatment is initiated. RNA is isolated from apatient's blood or a liver tissue sample and blotted as described above.An identical sample of blood or tissue is treated with antisenseoligonucleotide to inhibit HCV prior to RNA extraction and blotting. Theintensity of putative HCV signal in the two blots is then compared. Ifthe oligonucleotide is capable of inhibiting RNA function of thepatient-derived virus, the HCV signal will be reduced in theoligonucleotide-treated sample compared to the untreated sample. Thisindicates that the patient's HCV infection is responsive to treatmentwith the antisense oligonucleotide, and a course of therapeutictreatment can be initiated. If the two samples have identical signalsthe oligonucleotide is not able to inhibit replication of the virus, andanother method of treatment is indicated. Similar assays can be designedwhich employ other methods such as RT-PCR for RNA detection andquantitation, or Western blotting or ELISA for quantitation of HCV coreprotein expression, all of which are routinely performed by those in theart.

Example 9 The VHCV-IRES vaccinia/HCV Recombinant Virus Infected MouseModel

pSC11 (licensed from NIH) is a vaccinia virus expression vector thatuses vaccinia early and late promotor P7.5 to express foreign genes, andvaccinia late promotor P11 to express a LacZ gene. The vaccinia viralthymidine kinase (TK) sequence flanked these two promoter-expression DNAarrangements for homologous recombination. HCV RNA nucleotides 1-1357,including the HCV 5′ noncoding region, core and part of E1, obtainedfrom pHCV3, a cDNA clone from a chronic HCV patient with HCV type Hinfection, was fused to the 5′ end of a luciferase gene containing aSV40 polyadenylation signal sequence (Promega, pGL-2 promoter vector).The fused DNA fragment was placed under vaccinia promoter P7.5 of pSC11.The resultant construct was named pVNCELUA. A deletion of HCV RNAnucleotides 709 to 1357 was made in pVNCELUA and religation yielded theconstruct pVHCV-IRES. This construct uses the HCV initiator with theinternal ribosome entry initiating mechanism for translation. pVC-LUA isa luciferase control virus construct in which the luciferase geneincluding the translation initiation codon and polyadenylation signalwas directly placed under the P71.5 promoter of pSC11.

The basic experimental procedures for generating recombinant vacciniavirus by homologous recombination are known in the art. CV-1 cells forhomologous recombination and viral plaque and Hu TK-143B for TK-selection were purchased from the ATCC. Plasmid DNA transfection wasdone using lipofectin (GIBCO BRL). The selection of recombinant viruswas done by selection of viral plaques resistant to BrdU anddemonstrating luciferase and β-galactosidase activity. The virus waspurified through three rounds of plaque selection and used to prepare a100% pure viral stock. The virus-containing BSC-40 cells were harvestedin DMEM with 0.5% FBS followed by freeze-thawing three times todissociate the virus. Cellular debris was centrifuged out and thesupernatant was used for viral infection. A capital “V” was given to thename of each recombinant virus to distinguish it from the correspondingDNA construct (named with “p”)

Six-week old female Balb/c mice were purchased from Charles RiverLaboratories (Boston Mass.) . The mice were randomly grouped and werepretreated with oligonucleotide given subcutaneously once daily for twodays before virus infection and post-treated once at 4 hours afterinfection. The infection was carried out by intraperitoneal injection of1×10⁸ pfu of virus in 0.5 ml saline solution. At 24 hours afterinfection the liver was taken from each mouse and kept on dry ice untilit was homogenized at 30,000 rpm for about 30 seconds in 20 μl/mgluciferase reporter lysis buffer (Promega) using a Tissue Tearor(Biospec Products Inc.). Samples were transferred to eppendorf tubes onice and shaken by vortex for 20 seconds followed by centrifuging at 4°C. for 3 minutes 20 μl of supernatant was transferred to a 96-wellmicrotiter plate and 100 μl Luciferase Assay Reagent (Promega) wasadded. Immediately thereafter, the relative light units emitted weremeasured using a luminometer (ML 1000/Model 2.4, Dynatech Laboratories,Inc.).

Example 10 Evaluation of the 330 oligonucleotide ISIS 6547 in theVCHV-IRES infected mouse model:

A 20 mer deoxy oligonucleotide (the “330 oligonucleotide, ” SEQ ID NO: 6) targeted to nucleotides 330-349 surrounding the HCV translationinitiation codon has been shown in previous examples to specificallyinhibit HCV core protein synthesis in an in vitro translation assay,when tested as a phosphodiester. The phosphorothioatedeoxyoligonucleotide of the same sequence, ISIS 6547 demonstrated atleast a 50% reduction of HCV RNA when administered at dose of 100 nM totransformed human hepatocytes expressing HCV 5′ noncoding region, core,and part of the E1 product (nucleotides 1-1357 of HCV). Hanecak et al.,J. Virol. 1996, 70, 5203-5212. This effect was dose-dependent andsequence-dependent.

ISIS 6547 was evaluated in vivo using the VHCV-IRES infected mousemodel. Eight female Balb/c mice were pretreated subcutaneously witholigonucleotide in saline once daily for two days, then infectedintraperitoneally with 1×10⁸ pfu VHCV-IRES followed by a post-treatmentwith oligonucleotide four hours after infection. A group treated withsaline and infected with the same amount of VHCV-IRES served ascontrols. The effect of oligonucleotide on HCV gene expression wasmeasured by luciferase activity at 24 hours after infection. Whencompared to luciferase activity from VHCV-IRES-infected butsaline-treated controls, ISIS 6547 reduced luciferase signal in adose-dependent manner, giving 10.5% inhibition at 2 mg/kg, 28.2%inhibition at 6 mg/kg and 51.9% inhibition at 20 mg/kg. In contrast, theunrelated control oligonucleotide ISIS 1082 (GCCGAGGTCCATGTCGTACGC; SEQID NO. 36) exhibited no inhibitory effect at lower doses, thoughnon-specific inhibition of luciferase signal was observed at 20 mg/kg.Various routes of administration of oligonucleotide 6547 (subcutaneous,intravenous or intraperitoneal) gave similar levels of inhibition (76%,63% and 58%, respectively, at 20 mg/kg).

Example 11 Evaluation of 5-methyl-C modified 330 oligonucleotide, ISIS14803 in the VCHV-IRES infected mouse model

One of the heterocyclic base modifications presently available is5-methylcytosine (5-me-C) in which the nucleobase cytosine is methylatedat the 5-position. The corresponding nucleotide is 5-methylcytidine.Oligonucleotides containing this modification demonstrate higher targetbinding affinity than analogs without the base modification, and aresubstrates for RNAse H. Dean and Griffey, Antisense and Nucleic AcidDrug Development 1997, 7, 229-233. They also elicit less immunestimulation and complement stimulation than unmodified versions. Henryet al., Anti-Cancer Drug Design 1997, 12, 409-412.

A 5-me-C version of ISIS 6547 was synthesized in which every cytidinenucleotide was replaced by a 5-methylcytidine. This oligonucleotide,ISIS 14803, was evaluated in the VHCV-IRES system in mice, in directcomparison to its parent compound, ISIS 6547. Eight female Balb/c micewere subcutaneously treated with oligonucleotide in saline at one dayand two hours before infection and again at 4 hours after infection.Mice were infected by intraperitoneal injection with 1×10⁸ pfu per mouseof VHCV-IRES or VC-LUA. At 24 hours after infection, luciferase activityin liver was determined and compared to luciferase activity in livers ofa group of mice treated with saline and infected with the same amount ofVHCV-IRES or VC-LUA. ISIS 14803 showed 11.1% inhibition of liverluciferase activity at 2 mg/kg, 33.5% inhibition at 6 mg/kg and 59.1%inhibition at 20 mg/kg. ISIS 14803 did not show any inhibition ofluciferase activity in the control VC-LUA virus at the lower doses,though some nonspecific inhibition of luciferase activity was observedat the high dose of 20 mg/kg. Because this nonspecific inhibition wasalso observed with the control oligonucleotide, ISIS 1082, it wasthought to be a general class effect of high doses of phosphorothioateoligonucleotides.

37 20 nucleic acid Single Linear Yes unknown 1 TGCACGGTCT ACGAGACCTC 2020 nucleic acid Single Linear Yes unknown 2 GGTGCACGGT CTACGAGACC 20 20nucleic acid Single Linear Yes unknown 3 ATGGTGCACG GTCTACGAGA 20 20nucleic acid Single Linear Yes unknown 4 TCATGGTGCA CGGTCTACGA 20 20nucleic acid Single Linear Yes unknown 5 GCTCATGGTG CACGGTCTAC 20 20nucleic acid Single Linear Yes unknown 6 GTGCTCATGG TGCACGGTCT 20 20nucleic acid Single Linear Yes unknown 7 TCGTGCTCAT GGTGCACGGT 20 20nucleic acid Single Linear Yes unknown 8 ATTCGTGCTC ATGGTGCACG 20 20nucleic acid Single Linear Yes unknown 9 GGATTCGTGC TCATGGTGCA 20 20nucleic acid Single Linear Yes unknown 10 TAGGATTCGT GCTCATGGTG 20 20nucleic acid Single Linear Yes unknown 11 TTTAGGATTC GTGCTCATGG 20 20nucleic acid Single Linear Yes unknown 12 GGTTTAGGAT TCGTGCTCAT 20 20nucleic acid Single Linear Yes unknown 13 GAGGTTTAGG ATTCGTGCTC 20 20nucleic acid Single Linear Yes unknown modified base denoted as N isinosine 14 GAGGTTTAGG ATTNGTGCTC 20 20 nucleic acid Single Linear Yesunknown modified base denoted as N is inosine 15 GNGGTTTNGG ATTNGTGCTC20 20 nucleic acid Single Linear Yes unknown modified base denoted by Nis inosine 16 GNGGTTTNGG ANNNGTGCTC 20 20 nucleic acid Single Linear Yesunknown 17 TTGAGGTTTA GGATTCGTGC 20 20 nucleic acid Single Linear Yesunknown 18 CTTTGAGGTT TAGGATTCGT 20 20 nucleic acid Single Linear Yesunknown 19 TTCTTTGAGG TTTAGGATTC 20 20 nucleic acid Single Linear Yesunknown 20 TTTTCTTTGA GGTTTAGGAT 20 20 nucleic acid Single Linear Yesunknown 21 GTTTTTCTTT GAGGTTTAGG 20 20 nucleic acid Single Linear Yesunknown 22 TGGTTTTTCT TTGAGGTTTA 20 20 nucleic acid Single Linear Yesunknown 23 TTTGGTTTTT CTTTGAGGTT 20 20 nucleic acid Single Linear Yesunknown 24 CGTTTGGTTT TTCTTTGAGG 20 18 nucleic acid Single Linear Yesunknown 25 GTGCTCATGG TGCACGGT 18 17 nucleic acid Single Linear Yesunknown 26 GTGCTCATGG TGCACGG 17 16 nucleic acid Single Linear Yesunknown 27 GTGCTCATGG TGCACG 16 15 nucleic acid Single Linear Yesunknown 28 GTGCTCATGG TGCAC 15 10 nucleic acid Single Linear Yes unknown29 GTGCTCATGG 10 18 nucleic acid Single Linear Yes unknown 30 GCTCATGGTGCACGGTCT 18 17 nucleic acid Single Linear Yes unknown 31 CTCATGGTGCACGGTCT 17 15 nucleic acid Single Linear Yes unknown 32 CATGGTGCAC GGTCT15 10 nucleic acid Single Linear Yes unknown 33 TGCACGGTCT 10 18 nucleicacid Single Linear Yes unknown 34 TGCTCATGGT GCACGGTC 18 16 nucleic acidSingle Linear Yes unknown 35 GCTCATGGTG CACGGT 16 21 nucleic acid SingleLinear Yes unknown 36 GCCGAGGTCC ATGTCGTACG C 21 686 nucleic acid SingleLinear No unknown 37 GCCAGCCCCC GAUUGGGGGC GACACUCCAC CAUAGAUCACUCCCCUGUGA 50 GGAACUACUG UCUUCACGCA GAAAGCGUCU AGCCAUGGCG UUAGUAUGAG 100UGUCGUGCAG CCUCCAGGAC CCCCCCUCCC GGGAGAGCCA UAGUGGUCUG 150 CGGAACCGGUGAGUACACCG GAAUUGCCAG GACGACCGGG UCCUUUCUUG 200 GAUCAACCCG CTCAAUGCCUGGAGAUUUGG GCGUGCCCCC GCGAGACUGC 250 UAGCCGAGUA GUGUUGGGUC GCGAAAGGCCUUGUGGUACU GCCUGAUAGG 300 GUGCUUGCGA GUGCCCCGGG AGGUCUCGUA GACCGUGCACCAUGAGCACG 350 AAUCCUAAAC CUCAAAGAAA AACCAAACGU AACACCAACC GCCGCCCACA400 GGAGGUCAAG UUCCCGGGCG GUGGUCAGAU CGUUGGUGGA GUUUACCUGU 450UGCCGCGCAG GGGCCCCAGG UUGGGUGUGC GCGCGAUCAG GAAGACUUCC 500 GAGCGGUCGCAACCCCGUGG AAGGCGACAG CCUAUCCCCA AGGCUCGCCG 550 GCCCGAGGGC AGGGCCUGGGCUCAGCCCGG GUAUCCUUGG CCCCUCUAUG 600 GCAAUGAGGG CAUGGGGUGG GCAGGAUGGCUCCUGUCACC CCGCGGCUCC 650 CGGCCUAGUU GGGGCCCCAC GGACCCCCGG CGUAGG 686

What is claimed is:
 1. An antisense oligonucleotide up to 50 nucleotides in length which comprises at least a 5-nucleotide portion of SEQ ID NO:6 and which inhibits the function of HCV genomic or messenger RNA.
 2. The antisense oligonucleotide of claim 1 which has at least one phosphorothioate backbone linkage.
 3. The antisense oligonucleotide of claim 1 wherein at least one cytidine nucleotide is a 5′-methycytidine.
 4. A composition comprising the antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.
 5. The antisense oligonucleotide of claim 2 wherein every backbone linkage is a phosphorothioate backbone linkage and every cytidine nucleotide is a 5′-methylcytidine.
 6. An antisense oligonucleotide comprising SEQ ID NO:6.
 7. The antisense oligonucleotide of claim 6 which has at least one phosphorothioate backbone linkage.
 8. The antisense oligonucleotide of claim 6 wherein at least one cytidine nucleotide is a 5-methylcytidine.
 9. A composition comprising the oligonucleotide of claim 6 and pharmaceutically acceptable carrier.
 10. A phosphorothioate oligodeoxynucleotide comprising SEQ ID NO:6 wherein every cytidine nucleotide is a 5′-methylcytidine.
 11. A composition comprising the oligodeoxynucleotide of claim 10 and a pharmaceutically acceptable carrier. 